Helicobacter pylori fermentation process

Abstract

The present invention relates to a new method for the growth of Helicobacter pylori and purification of the cytotoxin produced by H. pylori. In particular, H. pylori is cultured in a medium comprising more than 1 gl −1 of glucose to produce a vacuolating cytotoxin.

1 . A method for culturing  H. pylori  to produce a vacuolating cytotoxin, wherein  H. pylori  is cultured in a medium comprising more than 1 gl −1  of glucose.  
     
     
         2 . A method according to  claim 1  wherein the medium is Brucella Broth medium supplemented with glucose and blood products.  
     
     
         3 . A method according to  claim 1  wherein the medium is Brucella Broth medium supplemented with glucose and a cyclodextrin.  
     
     
         4 . A method according to any preceding claim wherein the glucose is supplied by multiple-shot or continuous feeding in a fed batch process.  
     
     
         5 . A method according to any preceding claim wherein the glucose concetration of the medium is maintained at between 2 and 6 gl −1  throughout the culture period.  
     
     
         6 . A method for producing  H. pylori  vacuolating cytotoxin comprising culturing  H. pylori  in a medium supplemented with glucose as described in any one of  claims 1  to  5 .  
     
     
         7 . A method according to  claim 6  further comprising purifying the cytotoxin by adsorbing it onto a cellulose sulphate matrix and subsequently eluting it using a gradient of salt concetrations.  
     
     
         8 . A method for the purification of  H. pylori  vacuolatinq cytotoxin which comprises the steps of: 
 a) treating the supernatant of an  H. pylori  fermentation in order to concentrate the proteins therein;    b) bringing the proteins into suspension in a buffer comprising a salt concentration equivalent to 100 mM NaCl;    c) adsorbing the proteins onto a cellulose sulphate column;    d) eluting the bound proteins from the column using a salt gradient equivalent to 0.1 to 1.5 M NaCl in a phosphate buffer at pH 6.5;    e) selecting the fraction of the eluate which contains the vacuolating cytotoxin;    f) optionally, concentrating the cytotoxin further and subjecting it to size separation using a controlled pore matrix.    
     
     
         9 . A method according to  claim 8 , wherein step a) comprises precipitation in ammonium sulphate.  
     
     
         10 . A method according to  claim 8 , wherein steps a) and b) comprise tangential filtration and diafiltration of the sample.  
     
     
         11 . A method according to  claim 7  wherein the cytotoxin purification is performed as described in any one of  claims 8  to  10 .  
     
     
         12 . Use of  H. pylori  vacuolating cytotoxin when prepared according to the method of any one  claims 1  to  11  in the preparation of a composition for use as a vaccine.  
     
     
         13 . A method for vaccinating an organism against  H. pylori  infection which comprises preparing a sample of  H. pylori  vacoulating cytotoxin as described in any one of  claims 1  to  11  and adminstering a composition comprising said cytotoxin to the organism.